The determination and characterization of a cannabinoid receptor from brain are reported. A biologically active bicyclic cannabinoid analgetic CP-55,940 was tntium-Iabeled to high specific activity. Conditions for binding to rat brain P2 membranes and synaptosomes were established. The pH optimum was between 7 and 8, and specific binding could be eliminated by heating the membranes to 60#{176}. Binding to the P2 membranes was linear within the range of 10 to 50 g of protein/mi. Specific binding (defined as total binding displaced by 1 M 9-tetrahydrocannabinol(9 THC) or 100 nM desacetyllevonantradol) was saturable. The Kd determined from Scatchard analysis was 133 pM, and the Bmax for rat cortical P2 membranes was 1.85 pmol/mg of protein. The Hill coefficient for [3H] CP-55,940 approximated 1, indicating that, under the conditions of assay, a single class of binding sites was determined that did not exhibit cooperativity. The binding was rapid (k00 2.6 x 1 0 pM min) and reversible(K 0.016 min) and (k06’> 0.06 min). The two Kd values estimated from the kinetic constants approximately 55 M and exceeded 200 pM, respectively. The binding of the agonist ligand [3H] CP-55,940 was decreased by the nonhydrolyzable GTP analog guanylylimidodiphosphate. The guanine nucleotide induced a more rapid dissociation of the ligand from the binding site, consistent with an allostenc regulation of the putative receptor by a G protein. The binding was also sensitive to MgCI2 and CaCI2. Binding of [3H] CP-55,940 was displaced by cannabinoid drugs in the following order of potency: CP-55,940 desacetyllevonantradol> 11-OH-9-THC= 9-THC> cannabinol. Cannabidiol and cannabigerol displaced[3H] CP-55,940 by less than 50% at 1 M con-centrations. The (-)-isomer of CP-55,940 displaced with 50-fold greater potency than the (+)-isomer. This pharmacology is comparable to both the inhibition of adenylate cyclase in vitro and the analgetic activity of these compounds in vivo. The criteria for a high affinity, stereoselective, pharmacologically distinct cannabinoid receptor in brain tissue have been fulfilled.

Various preparations of Cannabis sativa (marihuana) have traditionally been used therapeutically and for their psychological manifestations[see reviews by Hollister(1) and Dewey (2)].\9-THC is the major compound in extracts of cannabis to have effects on the CNS(3). The predominant CNS responses to\9-THC include analgesia and antiemesis, as well as a “psychological high,” drowsiness, alterations in cognition and memory, and a decrement in psychomotor performance in humans(1, 2). Animal behavioral patterns associated with cannabinoid drug actions include altered behavior in monkeys, a characteristic static ataxia in dogs, and hypothermia, anal-